Project Summary/Abstract Chronic pain and itch (pruritis) drastically impact every aspect of daily function, often hindering work performance and severely impairing quality of life. Current treatments aimed at alleviating these conditions have serious side effects and lack sufficient efficacy for long-term use. Characterizing the molecular mechanisms by which noxious stimuli are processed may reveal candidate molecules to target with novel pharmacological treatments. Pain- and itch-sensing neurons whose cell bodies lie in the dorsal root ganglia (DRG) detect algogenic (pain-causing) and pruritogenic (itch-causing) stimuli in peripheral tissues and transmit the signal to the spinal cord, then to the brain. Signaling mediators released after injury or in the context of skin disorders both activate DRG neurons, leading to the perception of pain and itch, and sensitize these neurons, causing enhanced responses upon further stimulation. The signaling molecules that contribute to this sensitization activate receptors that yield diacylglycerol (DAG) production in the DRG. DAG can be converted into monoacylglycerol (MAG). DAG and MAG affect activity of DRG neurons by directly modulating receptor activity, activating secondary messengers that alter signaling, and acting as precursors to downstream metabolites that affect pain and itch. I have discovered that diacylglycerol kinase (DGK) can phosphorylate MAG in addition to phosphorylating DAG. Altering levels of DAG, MAG, or their metabolites has been shown to cause differences in pain signaling. Therefore, I am interested in determining if DGK?s phosphorylation of DAG and MAG can alter lipid levels in peripheral sensory neurons and lead to changes in itch and pain. I am investigating the DGK isoform iota (DGK?) that I found to be highly expressed in the pain- and itch-sensing DRG neurons of mice and humans. I hypothesize that DGK? kinase activity regulates levels of signaling lipids in DRG neurons, mediating somatosensory signaling. Using a DGK? knockout mouse, I have confirmed that kinase activity on both DAG and MAG is reduced in DRG neurons. Preliminary experiments with these mice show that sensitivity to pruritogens is enhanced by the loss of DGK?. To determine how alterations in lipid levels contributes to somatosensory phenotypes, I will use mass spectrometry to compare levels of DAG, MAG, and related signaling lipids in neuronal tissue between wild type and DGK?-/- mice. I will further examine sensitivity to itch in vivo using acute exposure to pruritogens. Additionally, I will examine pain sensitivity to noxious mechanical and thermal stimuli in DGK? knockout mice. Mice will be sensitized via inflammatory and neuropathic injury, and the severity of sensitization and recovery will be measured. Furthermore, I will investigate calcium responses to algogenic and pruritogenic stimuli that activate receptors regulated by these lipid signaling molecules in the DRG. In vitro signaling experiments and in vivo behavioral assays coupled with lipidomic analyses will help me characterize the role of DGK? in somatosensation and discover the potential of this kinase as targets for pain and itch therapies.